CN111549048A - Expression plasmid for bacterial asd gene balance lethal system and application thereof - Google Patents
Expression plasmid for bacterial asd gene balance lethal system and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of genetic engineering, and discloses an expression plasmid for a bacterial asd gene balanced lethal system and application thereof, wherein the expression plasmid comprises a Trc promoter, a multiple cloning site, an rrnB T1 terminator, an rrnB T2 terminator, a bom region, an ori replication initiation site, β lactamase andasda gene; green fluorescent protein is expressed in attenuated salmonella through the steps of gfp gene amplification, pEASY-blunt-gfp construction, pCW1910-gfp construction, HX delta asd strain conversion from pCW1910-gfp and the like. The expression plasmid provided by the invention removes the resistance gene, and solves the problemResistance genes have problems with gene drift in nature and green fluorescent protein is expressed in attenuated salmonella. The expression plasmids of the invention are suitable for use in a balanced lethal system in combination with a bacterial asd gene.
Description
Technical Field
The invention belongs to the technical field of biological genetic engineering, and relates to a plasmid vector, in particular to an expression plasmid for a bacterial asd gene balance lethal system and application thereof.
Background
Salmonella is a common pathogen causing food-borne diseases, and can cause human infection through polluted poultry meat and egg products, and the infection rate is high, thereby causing serious public health problems. The salmonella can also infect birds of different species and different ages in days, can cause acute or chronic infectious diseases, and causes huge economic loss to the poultry farming industry.
Salmonella invasive intracellular bacteria can directly present exogenous plasmid DNA to Antigen Presenting Cells (APC), and bacteria carrying the plasmid DNA dissolve in the APC or enter cytoplasm by host phagocytes and release the DNA to cell nucleus, so that antigen genes are expressed in vivo, and corresponding cellular immunity and humoral immunity reactions are induced, which is proved to be a good carrier carrying exogenous genes.
In recent years, people have attracted more and more interest in the way of weakening the virulence of salmonella through genetic engineering technology and further taking the attenuated salmonella as a vaccine or a vaccine live carrier. After the salmonella is attenuated by a genetic engineering method, the pathogenicity of the salmonella to a host is greatly reduced, and good invasiveness and immunogenicity can be still kept. Therefore, after entering the body, the vaccine using attenuated salmonella as a vector can still invade intestinal epithelial cells and colonize intestinal lymphoid tissues, and meanwhile, phagocytes continuously take up antigens to cause the body to generate corresponding immune response.
The current method for preparing vaccines by using attenuated salmonella generally comprises the following steps: the expression of foreign genes is obtained by cloning genes encoding a protein into plasmid vectors, which are then transformed into an attenuated strain of Salmonella. Unfortunately, the recombinant Salmonella obtained by this method has a low expression level of the foreign antigen, and another problem frequently encountered is unstable expression of the foreign gene in vivo. Meanwhile, the recombinant attenuated salmonella obtained by the method usually adopts an expression plasmid carrying a resistance gene to be used as a main genetic marker for the growth of the recombinant salmonella in vivo. However, because of the problem of gene drift in nature in the resistance gene, and in recent years, such plasmids containing resistance selection systems have not been accepted anymore due to biosafety considerations.
Disclosure of Invention
The invention aims to provide an expression plasmid for a bacterial asd gene balance lethal system, which removes resistance genes and solves the problem of gene drift of the resistance genes in nature;
another object of the present invention is to provide an application of the above expression plasmid for bacterial asd gene balanced lethal system.
In order to achieve the purpose, the technical method comprises the following steps:
an expression plasmid for a balanced lethal system of bacterial asd genes is pCW1910 plasmid, which comprises a Trc promoter, a multiple cloning site, a rrnB T1 terminator, a rrnB T2 terminator, a bom region, an ori replication initiation site, β lactamase andasda gene; the nucleotide sequence of pCW1910 plasmid is
AATGAGCTGTTGACAATTAATCATCCGGCTCGTATAATGTGTGGGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGACCATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGAATTCGCAATTCCCGGGGATCCGTCGACCTGCAGCCAAGCTCCCAAGCTTTTGGCTGTTTTGGCGGATGAGAGAAGATTTTCAGCCTGATACAGATTAAATCAGAACGCAGAAGCGGTCTGATAAAACAGAATTTGCCTGGCGGCAGTAGCGCGGTGGTCCCACCTGACCCCATGCCGAACTCAGAAGTGAAACGCCGTAGCGCCGATGGTAGTGTGGGGTCTCCCCATGCGAGAGTAGGGAACTGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATCCGCCGGGAGCGGATTTGAACGTTGCGAAGCAACGGCCCGGAGGGTGGCGGGCAGGACGCCCGCCATAAACTGCCAGGCATCAAATTAAGCAGAAGGCCATCCTGACGGATGGCCTTTTTGCGTTTCTCACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCGCAGCCATGACCCAGTCACGTAGCGATAGCGGAGTGTATACTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGACTAGTTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTGCAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAACACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGACTAGTCTACGCCAACTGGCGCAGCATTCGACGCAGCGGCTCGGCGGCGCCCCATAACAACTGGTCGCCTACGGTAAACGCCGACAAGAACTCTGGCCCCATGTTCAGCTTACGCAGACGACCAACCGGCGTAGTCAACGTGCCGGTCACCGCCGCCGGGGTTAATTCGCGCATAGTGATATCACGATCGTTCGGCACCACTTTCGCCCACGGATTATGTGCCGCCAGCAGTTCTTCCACCGTCGGAATGGATACCTCTTTTTTCAGCTTGATGGTGAACGCCTGGCTGTGACAGCGCAGCGCGCCGACGCGCACGCACAAACCATCAACCGGAATCACAGAGGCAGTATTGAGAATCTTGTTGGTTTCCGCCTGGCCTTTCCACTCTTCGCGGCTCTGGCCGTTATCGAGCTGTTTGTCGATCCAGGGGATCAGGCTTCCCGCCAGCGGTACGCCAAAGTTATCAACCGGCAGCTCGCCGCTGCGGGTCAATGCCGTAACTTTGCGTTCAATATCAAGAATTGCGGAAGACGGCGTCGCCAGTTCATCGGCGACATGGCCATACAACTGACCCATCTGGGTTAACAGCTCGCGCATATGGCGCGCGCCGCCGCCGGAGGCGGCCTGATAGGTCGCGACGGATACCCAGTCAACGAGATTATGGGCAAAGAGACCGCCCAGCGACATCAACATCAGGCTAACGGTACAGTTACCGCCCACAAAGGTCTTCACGCCATTGTTCAGGCCGTCGGTAATCACGTCCTGGTTAACCGGGTCGAGAATAATAATGGCATCATCTTTCATGCGCAGCGTAGAAGCCGCGTCAATCCAGTAACCCTGCCATCCGCTTTCGCGCAGCTTTGGATAAATTTCGTTGGTATAATCGCCGCCCTGGCAGGTCACGATGATATCGAGCGCTTTTAGCGCATCCAGATCAAAAGCGTCCTGTAGCGTGCCGGTGGAGGTGTCGCCGAAGGTGGGCGCCGCCTGTCCAAACTGGGAGGTAGAAAAGAAAACAGGGCGAATAGCGTCGAAATCGCGCTCCTCTACCATGCGTTGCATGAGAACAGAGCCGACCATTCCGCGCCAGCCGATAAAACCAACATTTTTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCG。
As a limitation: the nucleotide sequence of the Trc promoter is 10 th to 39 th positions of the nucleotide sequence of the pCW1910 plasmid.
As another limitation: the nucleotide sequence of the rrnB T1 terminator is 448-534 th site of the nucleotide sequence of the pCW1910 plasmid; the nucleotide sequence of the rrnB T2 terminator is 626-653 th of the nucleotide sequence of the pCW1910 plasmid.
As a third limitation: the nucleotide sequence of the bom region is 764-904 of the nucleotide sequence of the pCW1910 plasmid.
As a fourth limitation: the nucleotide sequence of the ori replication initiation site is 1090-1678 th site of the nucleotide sequence of pCW 1910.
As a fifth limitation, the β lactamase gene has two endsSpeI restriction endonuclease site, the nucleotide sequence of which is 1849-2826 of the nucleotide sequence of pCW1910 plasmid.
As a further limitation: the nucleotide sequence of the asd gene is 2827-3933 of the nucleotide sequence of the pCW1910 plasmid.
The invention also provides an application of the expression plasmid for the bacterial asd gene balance lethal system, which is applied to the attenuated salmonella for expressing green fluorescent protein.
As a limitation: the method for expressing green fluorescent protein by applying pCW1910 plasmid to attenuated salmonella is carried out according to the following steps:
(I) gfp Gene amplification
Taking a plasmid containing a GFP gene as a template, taking GFP-F and GFP-R as primers, amplifying the GFP gene by adopting a PCR technology to obtain a PCR product, detecting the PCR product by using agarose gel electrophoresis, cutting a target strip, and purifying by using a gel recovery kit to obtain a GFP gene fragment;
GFP-F：5-AAGCTTATGGAGAGCGACGAGAGC-3
GFP-R：5-AAGCTTTCAGCGAGATCCGGTGGAGC-3
(II) pEASY-blunt-gfp construction
Connecting the gfp gene fragment to a pEASY-blunt cloning vector;
(III) construction of pCW1910-gfp
Using restriction endonuclease Hind III to cut pEASY-blunt-gfp and pCW1910 plasmid, recovering gfp gene fragment and pCW1910 plasmid, and connecting;
(IV) HX.DELTA.asd Strain construction
Preparing HX strain competent cells by using single HX strain colonies, adding pKD46 plasmid for transformation, screening HX/pKD46 positive colonies, performing electrotransfer by using pKD4 plasmid as a template and asd-F and asd-R as primers, performing PCR amplification on purified targeting sequences, eliminating pKD46 plasmid after screening positive colonies, performing electrotransfer by using pCP20 plasmid to eliminate pCP20 plasmid, and detecting to obtain HX delta asd strain;
(V) pCW1910-gfp HX.DELTA.asd strain transformation
And transforming the pCW1910 plasmid connected with the gfp gene into an HX delta asd strain to obtain the HX delta asd/pCW1910-gfp strain.
The HX delta asd/pCW1910-gfp strain of the invention is classified and named as salmonella pullorum, the preservation number is CGMCC No.19579, the strain is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, and the preservation time is 2020, 04, 15 days; the HX delta asd strain is classified and named as Salmonella pullorum with the preservation number of CGMCC No.19578, and is preserved in the China general microbiological culture Collection center of China Committee for culture Collection of microorganisms for 2020 and 04, 15 days.
Due to the adoption of the scheme, compared with the prior art, the invention has the beneficial effects that:
(1) the expression plasmid for the bacterial asd gene balance lethal system provided by the invention removes the resistance gene, and solves the problem of gene drift of the resistance gene in nature;
(2) salmonella used in the present inventionasdThe gene has higher conservative rate and can be matched with salmonella of different serotypesasdThe gene-deleted strain is combined into a host-vector lethal balance system, overcomes various adverse effects caused by the conventional vector vaccine, and has wide application range.
The expression plasmids of the invention are suitable for use in a balanced lethal system in combination with a bacterial asd gene.
Drawings
The invention is described in further detail below with reference to the figures and the embodiments.
FIG. 1 is a map of pCW1910 plasmid according to an embodiment of the present invention;
FIG. 2 shows PCR detection of positive clones after electroporation of pKD46 plasmid into HX strain according to an embodiment of the present invention;
FIG. 3 shows an embodiment of the present inventionasdRecovering the targeting gene and then carrying out electrophoresis detection;
FIG. 4 shows the colony PCR assay after the targeting gene fragment of the embodiment of the invention is transformed into HX/pKD 46;
FIG. 5 shows the elimination assay of pKD46 plasmid according to an embodiment of the present invention;
FIG. 6 shows the transformation of plasmid pCP20 into HX.DELTA.asd∷kanDetecting the result;
FIG. 7 shows the result of plasmid elimination of pCP20 in accordance with the present invention;
FIG. 8 shows the present invention example of the strain after elimination of pCP20 plasmidasdDetecting genes;
FIG. 9 shows growth curves of HX Δ asd strain, HX Δ asd/pCW1910-gfp strain and HX strain according to examples of the present invention;
FIG. 10 shows SDS-PAGE results of the present invention.
Detailed Description
The present invention is further described with reference to the following examples, but it should be understood by those skilled in the art that the present invention is not limited to the following examples, and any modifications and equivalent changes based on the specific examples of the present invention are within the scope of the claims of the present invention.
Example expression plasmid for bacterial asd Gene Balanced lethal System and use thereof
An expression plasmid for the balanced lethal system of bacterial asd gene of this example is pCW1910 plasmid, which is artificially synthesized by a chemical method of whole gene synthesis, and its map is shown in FIG. 1, and comprises Trc promoter, multiple cloning site, rrnB T1 terminator, rrnB T2 terminator, bom region, ori replication initiation site, β lactamase and lactamaseasdA gene. Wherein:
the nucleotide sequence of the Trc promoter is:
5’-TTGACAATTAATCATCCGGCTCGTATAATG-3’；
the nucleotide sequence of rrnB T1 terminator is:
5’-CAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAAT-3’;
the nucleotide sequence of rrnB T2 terminator is:
5’-AGAAGGCCATCCTGACGGATGGCCTTTT-3’；
the nucleotide sequence of the bom region is:
CGCAGCCATGACCCAGTCACGTAGCGATAGCGGAGTGTATACTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGG；
the nucleotide sequence of ori replication origin is:
TTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAA；
β the end and the tail of the lactamase haveSpeI restriction endonuclease site, the nucleotide sequence of which is:
ACTAGTTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTGCAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAACACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGACTAGT；
the nucleotide sequence of the asd gene is:
CTACGCCAACTGGCGCAGCATTCGACGCAGCGGCTCGGCGGCGCCCCATAACAACTGGTCGCCTACGGTAAACGCCGACAAGAACTCTGGCCCCATGTTCAGCTTACGCAGACGACCAACCGGCGTAGTCAACGTGCCGGTCACCGCCGCCGGGGTTAATTCGCGCATAGTGATATCACGATCGTTCGGCACCACTTTCGCCCACGGATTATGTGCCGCCAGCAGTTCTTCCACCGTCGGAATGGATACCTCTTTTTTCAGCTTGATGGTGAACGCCTGGCTGTGACAGCGCAGCGCGCCGACGCGCACGCACAAACCATCAACCGGAATCACAGAGGCAGTATTGAGAATCTTGTTGGTTTCCGCCTGGCCTTTCCACTCTTCGCGGCTCTGGCCGTTATCGAGCTGTTTGTCGATCCAGGGGATCAGGCTTCCCGCCAGCGGTACGCCAAAGTTATCAACCGGCAGCTCGCCGCTGCGGGTCAATGCCGTAACTTTGCGTTCAATATCAAGAATTGCGGAAGACGGCGTCGCCAGTTCATCGGCGACATGGCCATACAACTGACCCATCTGGGTTAACAGCTCGCGCATATGGCGCGCGCCGCCGCCGGAGGCGGCCTGATAGGTCGCGACGGATACCCAGTCAACGAGATTATGGGCAAAGAGACCGCCCAGCGACATCAACATCAGGCTAACGGTACAGTTACCGCCCACAAAGGTCTTCACGCCATTGTTCAGGCCGTCGGTAATCACGTCCTGGTTAACCGGGTCGAGAATAATAATGGCATCATCTTTCATGCGCAGCGTAGAAGCCGCGTCAATCCAGTAACCCTGCCATCCGCTTTCGCGCAGCTTTGGATAAATTTCGTTGGTATAATCGCCGCCCTGGCAGGTCACGATGATATCGAGCGCTTTTAGCGCATCCAGATCAAAAGCGTCCTGTAGCGTGCCGGTGGAGGTGTCGCCGAAGGTGGGCGCCGCCTGTCCAAACTGGGAGGTAGAAAAGAAAACAGGGCGAATAGCGTCGAAATCGCGCTCCTCTACCATGCGTTGCATGAGAACAGAGCCGACCATTCCGCGCCAGCCGATAAAACCAACATTTTTCAT；
the nucleotide sequence of the pCW1910 plasmid is:
AATGAGCTGTTGACAATTAATCATCCGGCTCGTATAATGTGTGGGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGACCATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGAATTCGCAATTCCCGGGGATCCGTCGACCTGCAGCCAAGCTCCCAAGCTTTTGGCTGTTTTGGCGGATGAGAGAAGATTTTCAGCCTGATACAGATTAAATCAGAACGCAGAAGCGGTCTGATAAAACAGAATTTGCCTGGCGGCAGTAGCGCGGTGGTCCCACCTGACCCCATGCCGAACTCAGAAGTGAAACGCCGTAGCGCCGATGGTAGTGTGGGGTCTCCCCATGCGAGAGTAGGGAACTGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATCCGCCGGGAGCGGATTTGAACGTTGCGAAGCAACGGCCCGGAGGGTGGCGGGCAGGACGCCCGCCATAAACTGCCAGGCATCAAATTAAGCAGAAGGCCATCCTGACGGATGGCCTTTTTGCGTTTCTCACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCGCAGCCATGACCCAGTCACGTAGCGATAGCGGAGTGTATACTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGACTAGTTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTGCAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAACACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGACTAGTCTACGCCAACTGGCGCAGCATTCGACGCAGCGGCTCGGCGGCGCCCCATAACAACTGGTCGCCTACGGTAAACGCCGACAAGAACTCTGGCCCCATGTTCAGCTTACGCAGACGACCAACCGGCGTAGTCAACGTGCCGGTCACCGCCGCCGGGGTTAATTCGCGCATAGTGATATCACGATCGTTCGGCACCACTTTCGCCCACGGATTATGTGCCGCCAGCAGTTCTTCCACCGTCGGAATGGATACCTCTTTTTTCAGCTTGATGGTGAACGCCTGGCTGTGACAGCGCAGCGCGCCGACGCGCACGCACAAACCATCAACCGGAATCACAGAGGCAGTATTGAGAATCTTGTTGGTTTCCGCCTGGCCTTTCCACTCTTCGCGGCTCTGGCCGTTATCGAGCTGTTTGTCGATCCAGGGGATCAGGCTTCCCGCCAGCGGTACGCCAAAGTTATCAACCGGCAGCTCGCCGCTGCGGGTCAATGCCGTAACTTTGCGTTCAATATCAAGAATTGCGGAAGACGGCGTCGCCAGTTCATCGGCGACATGGCCATACAACTGACCCATCTGGGTTAACAGCTCGCGCATATGGCGCGCGCCGCCGCCGGAGGCGGCCTGATAGGTCGCGACGGATACCCAGTCAACGAGATTATGGGCAAAGAGACCGCCCAGCGACATCAACATCAGGCTAACGGTACAGTTACCGCCCACAAAGGTCTTCACGCCATTGTTCAGGCCGTCGGTAATCACGTCCTGGTTAACCGGGTCGAGAATAATAATGGCATCATCTTTCATGCGCAGCGTAGAAGCCGCGTCAATCCAGTAACCCTGCCATCCGCTTTCGCGCAGCTTTGGATAAATTTCGTTGGTATAATCGCCGCCCTGGCAGGTCACGATGATATCGAGCGCTTTTAGCGCATCCAGATCAAAAGCGTCCTGTAGCGTGCCGGTGGAGGTGTCGCCGAAGGTGGGCGCCGCCTGTCCAAACTGGGAGGTAGAAAAGAAAACAGGGCGAATAGCGTCGAAATCGCGCTCCTCTACCATGCGTTGCATGAGAACAGAGCCGACCATTCCGCGCCAGCCGATAAAACCAACATTTTTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCG。
this example also includes the use of the above plasmid pCW1910 for the expression of green fluorescent protein in attenuated Salmonella in the following sequence of steps:
(I) gfp Gene amplification
Taking a plasmid containing a GFP gene as a template, taking GFP-F and GFP-R as primers, amplifying the GFP gene by adopting a PCR technology to obtain a PCR product, detecting the PCR product by using 1% agarose gel electrophoresis, cutting a target band, and purifying by using a gel recovery kit to obtain a GFP gene fragment;
GFP-F：5-AAGCTTATGGAGAGCGACGAGAGC-3
GFP-R：5-AAGCTTTCAGCGAGATCCGGTGGAGC-3
the PCR reaction system is as follows: 1 mu L of each primer GFP-F and GFP-R, 10 mu L of 2 XPCR Mix Buffer, 7 mu L of sterile water, 1 mu L of plasmid template containing GFP gene, and the total amount is 20 mu L;
the PCR reaction conditions are as follows: pre-denaturation at 94 ℃ for 5min, denaturation at 94 ℃ for 30s, annealing at 52 ℃ for 30s, extension at 72 ℃ for 60s, performing 30 cycles in the process from denaturation to extension, and finally extension at 72 ℃ for 5 min;
(II) pEASY-blunt-gfp construction
Connecting the gfp gene fragment to a pEASY-blunt cloning vector purchased from Beijing holotype gold organisms;
(III) construction of pCW1910-gfp
Using restriction endonuclease Hind III to cut pEASY-blunt-gfp and pCW1910 plasmid, recovering gfp gene fragment and pCW1910 plasmid, and connecting;
(IV) HX. DELTAasdStrain construction
1）asdGene knockout
Preparation of electrotransformation competent cells
Under aseptic condition, selecting single colony of HX strain, inoculating into LB liquid culture medium, culturing at 37 deg.C and 180rpm for 16h, inoculating into 20mL fresh LB at 1%, culturing at 37 deg.C and 180rpm, and culturing at OD600Stopping culturing when the concentration is 0.4-0.6, transferring the bacterial liquid to a sterile precooling centrifuge tube, centrifuging at 6000rpm for 15min at 4 ℃, collecting thalli, adding 10mL of precooled 10% glycerol, gently resuspending the thalli, centrifuging at 4 ℃ and 4000rpm for 5min, collecting the thalli, repeating the process for 3 times, collecting the thalli for the last time, adding 400 mu L of precooled 10% glycerol, gently resuspending the thalli, subpackaging according to 100 mu L per tube, quickly placing in liquid nitrogen for freezing, and then placing at-70 ℃ for later use;
(ii) transformation of pKD46 plasmid
Adding 2 mu L of pKD46 plasmid (about 20 ng) into prepared HX strain competent cells, uniformly mixing, transferring to a precooled electric shock cup, shocking at 1800V for 5ms, immediately adding 900 mu L of LB liquid culture medium preheated at 30 ℃, reviving at 30 ℃ for 1h at 180rpm, centrifuging at 6000rpm for 5min, removing 800 mu L of supernatant, re-suspending the thalli, completely coating the thalli on an LB plate (Amp +), and culturing at 30 ℃;
③ HX/pKD46 positive bacterial colony screening
Selecting a single colony to be detected on an LB plate, suspending the single colony in 10 mu L of sterile water, taking the bacterial liquid as a template, carrying out PCR detection by using a pKD46-jc-F/R sequence, taking the sterile water as the template as a negative control, taking a plasmid pKD46 as a positive control, and detecting a PCR product by using 1% agarose gel, wherein the detection result is shown in figure 2, the pKD46 plasmid is transferred into an HX strain, and M in the figure is 2 KMarker; 1-7 are randomly selected positive clones; plasmid pKD46, positive control, 8; 9 is a negative control;
pKD46-jc-F： 5’-CCGCAGAGCAGAAGGT-3’；
pKD46-jc-R： 5’-TCAGCAAGCAGGGTGT-3’；
amplification and purification of target sequence
PCR amplification was carried out using pKD4 plasmid as a template and asd-F and asd-R as primers. The reaction system is as follows: 1 μ L of each primer asd-F/R, 10 μ L of 2 XMix Buffer, 7 μ L of sterile water, 1 μ L of bacterial liquid template, and 20 μ L of the total. The PCR reaction conditions are as follows: pre-denaturation at 94 ℃ for 5min, denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, extension at 72 ℃ for 60s, 30 cycles of denaturation-extension processes, and final extension at 72 ℃ for 5 min. The PCR product was detected by 1% agarose gel electrophoresis, and the detection results are shown in FIG. 3, where the targeted gene fragment of the expected size is found between 1000-2000bp, M: 2K Marker; lanes 1 and 2 are the recovered targeted gene fragments;
asd-F：5’-AGGATACTGGCGCGCATACACAGCACATCTCTTTGCAGGAAAAAAACGCTGTGTAGGCTGGAGCTGCTTC-3’
asd-R：5’-TATCCGGCCTACAGAACCACACGCAGGCCCGATAAGCGCTGCAATAGCTATTAACGGCTGACATGGGAATTAG-3’
electric transfer of targeted gene fragment
Adding 3 μ of targeting gene fragment (about 200 ng) into HX/pKD46 positive colony, mixing, transferring to precooled electric shock cup, shock at 2500V for 5ms, immediately adding LB liquid culture medium preheated at 37 deg.C (800 μ L), activating at 37 deg.C and 180rpm for 1h, centrifuging at 4000rpm for 5min, removing 700 μ L of supernatant, re-suspending the thallus with the rest supernatant, and coating on LB plate (DAP)+，Kan+) Inversely culturing for 40h at 30 ℃;
sixth, PCR screening of positive strains
Picking LB plate (DAP)+，Kan+) The cultured colony is dissolved in 10 mu L of sterile water and then is used as a template, asd-JC-F and asd-JC-R are used as primers to carry out PCR amplification, the reaction system is that the primers asd-JC-F/R are 1 mu L each, 2 × Mix Buffer is 10 mu L, sterile water is 7 mu L, the bacterial liquid template is 1 mu L, and the overall system is 20 mu L, the PCR reaction conditions are that the primer is pre-denatured at 94 ℃ for 5min, denatured at 94 ℃ for 30s, annealed at 55 ℃ for 30s, extended at 72 ℃ for 30s, the process of denaturation to extension is carried out for 30 cycles, finally, the PCR product is extended at 72 ℃ for 5min, and is detected by using 1% gel electrophoresis, the detection result is shown in figure 4, the size of the amplified fragment of the agarose strain HX is 1188bp, which is negative, the size of the amplified fragment of a 4 lane is 1589bp, which is expected, namely a positive colony, wherein M is 2K Marker in the figure, 1 is HX, and 2-8 is a colony after transformation;
asd-jc-F：5’- GGCGCGCATA CACAGCAC-3’
asd-jc-R：5’-CTACAGAACCACACGCAGGC-3’
2) pKD46 plasmid elimination
PCR detection of the correct HX.DELTA.asd: kan/pKD 46 strain, inoculated on LB plate (DAP)+，Kan+) After culturing for 72h at 42 ℃, picking a single colony, detecting whether the plasmid is eliminated by a pKD46 detection primer, wherein the reaction system comprises 1 mu L of each primer pKD46-jc-F/R, 1 mu L of 2 × Mix Buffer 10 mu L, 7 mu L of sterile water, 1 mu L of a bacterial liquid template and 20 mu L of the overall system, PCR reaction conditions comprise pre-denaturation at 94 ℃ for 5min, denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, extension at 72 ℃ for 30s, and 30 cycles from the denaturation to the extension process, and finally extension at 72 ℃ for 5min, PCR products are detected by using 1% agarose gel electrophoresis, and the detection result is shown in figure 5, wherein a positive control (lane 1) shows a specific band between 100bp and 250bp, while a negative control (lane 2) and the picked colony (lanes 3-9) show no band, which shows that the pKD46 plasmid in the bacterium is successfully eliminated, and M is 2 KDa positive marker (539D 2) and a single colony (3-9) is a sterile water control;
3) pCP20 plasmid transformation of HX. DELTA.asd: kan strain
① HXΔasd∷kanStrain competenceCell preparation
Under aseptic conditions, picking HX deltaasd∷kanInoculating single colony into LB liquid culture medium containing DAP, culturing at 37 deg.C and 180rpm for 16h, inoculating to 20mL of fresh LB at 1%, culturing at 37 deg.C and 180rpm, and culturing at OD600Stopping culturing when the concentration is 0.4-0.6, transferring the bacterial liquid to a sterile precooling centrifuge tube, centrifuging at 6000rpm for 15min at 4 ℃, collecting thalli, adding 10mL of precooled 10% glycerol, gently resuspending the thalli, centrifuging at 4 ℃ and 4000rpm for 5min, collecting the thalli, repeating the process for 3 times, collecting the thalli for the last time, adding 400 mu L of precooled 10% glycerol, gently resuspending the thalli, subpackaging according to 100 mu L per tube, quickly placing in liquid nitrogen for freezing, and then placing at-70 ℃ for later use;
② pCP20 plasmid transformed HX deltaasd∷kanBacterial strains
80 μ L of prepared HX delta is takenasd∷kanStrain competent cells were mixed with 5. mu.L of pCP20 plasmid (about 30 ng), shocked at 1800V for 5ms, and 900. mu.L of LB liquid medium (DAP) was added+) Activating at 30 deg.C and 180rpm for 1 hr, centrifuging at 6000rpm for 5min, removing 700 μ L supernatant, resuspending the thallus with the rest culture medium, and coating on LB plate (Amp)+，DAP+) The reaction system comprises 1 mu L of each primer pCP20-F/R, 10 mu L of 2 × Mix Buffer, 7 mu L of sterile water and 1 mu L of bacterial liquid template, wherein the total amount is 20 mu L, the PCR reaction conditions comprise pre-denaturation at 94 ℃ for 5min, denaturation at 94 ℃ for 30s, annealing at 52 ℃ for 30s, extension at 72 ℃ for 30s, 30 cycles of denaturation-extension processes, and finally extension at 72 ℃ for 5min, PCR products are detected by using 1% agarose gel electrophoresis, the detection result is shown in figure 6, and specific bands with the same size as that of a positive control (lane 9) band exist between 100bp and 250bp of lanes 1-7, which indicates that the pCP20 plasmid is successfully transferred into HX deltaasdThe: kan strain, in the figure M: 2K DNA Marker; 1-8 are selected colonies; pCP20 plasmid (positive control) 9; 10 is sterile water (negative control);
pCP20-jc-F： 5’-GTGGCTTCTGTTTCTATC-3’；
pCP20-jc-R： 5’-ACCTGAGTCGCTGTCT-3’；
4) pCP20 plasmid Elimination
PCR detection of the correct HX.DELTA.asdthe/pCP 20 strain was inoculated on LB plates (DAP)+) Placing the mixture at 42 ℃ for static culture for 72h, selecting a single colony for PCR detection, wherein the reaction system comprises 1 mu L of each primer pCP20-jc-F/R, 10 mu L of 2 × Mix Buffer, 7 mu L of sterile water and 1 mu L of bacterial liquid template, the total system is 20 mu L, the PCR reaction conditions comprise pre-denaturation at 94 ℃ for 5min, denaturation at 94 ℃ for 30s, annealing at 52 ℃ for 30s, extension at 72 ℃ for 30s, 30 cycles of denaturation-extension process, and finally extension at 72 ℃ for 5min, PCR products are detected by using 1% agarose gel electrophoresis, the detection result is shown in figure 7, the colony eliminated by the plasmid has no specific band (lane 3), which shows that the pCP20 plasmid is successfully eliminated, M is 2K DNA Marker in the figure, 1 is pCP20 (positive control), 2 is sterile water (negative control), and 3 is HX delta 3asdStrain; then the bacterial strain with pCP20 plasmid eliminated is processedasdThe results of the gene detection are shown in FIG. 8, and the HX strain was amplifiedasdThe fragment size is 1188 bp; HX DeltaasdAmplification of: kan StrainasdThe fragment size is 1589bp, HX deltaasdPlant amplificationasdThe sizes of the fragments are 204bp, which are all in accordance with the expected sizes,asdthe gene is completely deleted, wherein M is 2K DNAmarker; 1 is HX strain; 2 is HX.DELTA.asdThe: kan strain; 3 is HX.DELTA.asdStrain;
(V) pCW1910-gfp HX.DELTA.asd strain transformation
And transforming the pCW1910 plasmid connected with the gfp gene into an HX delta asd strain to obtain the HX delta asd/pCW1910-gfp strain.
The growth states of the HX delta asd strain, the HX strain and the HX delta asd/pCW1910-gfp strain were detected to obtain a growth curve as shown in FIG. 9. As can be seen from FIG. 9, the HX delta asd strain could not grow normally, and the growth states of the HX delta asd/pCW1910-gfp strain and the HX strain are consistent, which indicates that the asd gene on the pCW1910 plasmid can be complementary to the HX delta asd strain.
Detection of GFP protein by polyacrylamide gel electrophoresis: taking 3ml of overnight cultured HX delta asd/pCW1910-gfp bacterial solution, centrifuging to collect bacteria, detecting by polyacrylamide gel electrophoresis, wherein the detection result is shown in figure 10, M is a protein Marker in figure 10, HX strain in lane 1 and HX delta in lane 2asdthe/pCW 1910 engineered bacterium, lanes 3 and 4 are both HX Δ asd/pCW1910-gfp engineered bacterium,the arrow in the figure indicates that GFP protein is present, indicating that pCW1910 plasmid can express the foreign gene GFP protein in attenuated Salmonella.
SEQUENCE LISTING
<110> Hebei Kogyo pharmaceutical Co., Ltd
<120> expression plasmid for bacterial asd gene balance lethal system and application thereof
<130>2020
<160>1
<170>PatentIn version 3.5
<210>1
<211>4038
<212>DNA
<213> Artificial sequence
<400>1
aatgagctgt tgacaattaa tcatccggct cgtataatgt gtgggtggaa ttgtgagcgg 60
ataacaattt cacacaggaa acagaccatg agtattcaac atttccgtgt cgcccttatt 120
cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta 180
aaagatgctg aagaattcgc aattcccggg gatccgtcga cctgcagcca agctcccaag 240
cttttggctg ttttggcgga tgagagaaga ttttcagcct gatacagatt aaatcagaac 300
gcagaagcgg tctgataaaa cagaatttgc ctggcggcag tagcgcggtg gtcccacctg 360
accccatgcc gaactcagaa gtgaaacgcc gtagcgccga tggtagtgtg gggtctcccc 420
atgcgagagt agggaactgc caggcatcaa ataaaacgaa aggctcagtc gaaagactgg 480
gcctttcgtt ttatctgttg tttgtcggtg aacgctctcc tgagtaggac aaatccgccg 540
ggagcggatt tgaacgttgc gaagcaacgg cccggagggt ggcgggcagg acgcccgcca 600
taaactgcca ggcatcaaat taagcagaag gccatcctga cggatggcct ttttgcgttt 660
ctcacatgca gctcccggag acggtcacag cttgtctgta agcggatgcc gggagcagac 720
aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg gggcgcagcc atgacccagt 780
cacgtagcga tagcggagtg tatactggct taactatgcg gcatcagagc agattgtact 840
gagagtgcac catatgcggt gtgaaatacc gcacagatgc gtaaggagaa aataccgcat 900
caggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg 960
agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc 1020
aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt 1080
gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag 1140
tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc 1200
cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc 1260
ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt 1320
cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt 1380
atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc 1440
agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa 1500
gtggtggcct aactacggct acactagaag gacagtattt ggtatctgcg ctctgctgaa 1560
gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg 1620
tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga 1680
agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg 1740
gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg 1800
aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagac tagtttacca 1860
atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc 1920
ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc 1980
tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc 2040
agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat 2100
taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt 2160
tgccattgct gcaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc 2220
cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag 2280
ctccttcggt cctccgatcg ttgtcagaag taagttggccgcagtgttat cactcatggt 2340
tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac 2400
tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg 2460
cccggcgtca acacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat 2520
tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc 2580
gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc 2640
tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa 2700
atgttgaata ctcatactct tcctttttca atattattga agcatttatc agggttattg 2760
tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg 2820
actagtctac gccaactggc gcagcattcg acgcagcggc tcggcggcgc cccataacaa 2880
ctggtcgcct acggtaaacg ccgacaagaa ctctggcccc atgttcagct tacgcagacg 2940
accaaccggc gtagtcaacg tgccggtcac cgccgccggg gttaattcgc gcatagtgat 3000
atcacgatcg ttcggcacca ctttcgccca cggattatgt gccgccagca gttcttccac 3060
cgtcggaatg gatacctctt ttttcagctt gatggtgaac gcctggctgt gacagcgcag 3120
cgcgccgacg cgcacgcaca aaccatcaac cggaatcaca gaggcagtat tgagaatctt 3180
gttggtttcc gcctggcctt tccactcttc gcggctctgg ccgttatcga gctgtttgtc 3240
gatccagggg atcaggcttc ccgccagcgg tacgccaaag ttatcaaccg gcagctcgcc 3300
gctgcgggtc aatgccgtaa ctttgcgttc aatatcaaga attgcggaag acggcgtcgc 3360
cagttcatcg gcgacatggc catacaactg acccatctgg gttaacagct cgcgcatatg 3420
gcgcgcgccg ccgccggagg cggcctgata ggtcgcgacg gatacccagt caacgagatt 3480
atgggcaaag agaccgccca gcgacatcaa catcaggcta acggtacagt taccgcccac 3540
aaaggtcttc acgccattgt tcaggccgtc ggtaatcacg tcctggttaa ccgggtcgag 3600
aataataatg gcatcatctt tcatgcgcag cgtagaagcc gcgtcaatcc agtaaccctg 3660
ccatccgctt tcgcgcagct ttggataaat ttcgttggta taatcgccgc cctggcaggt 3720
cacgatgata tcgagcgctt ttagcgcatc cagatcaaaa gcgtcctgta gcgtgccggt 3780
ggaggtgtcg ccgaaggtgg gcgccgcctg tccaaactgg gaggtagaaa agaaaacagg 3840
gcgaatagcg tcgaaatcgc gctcctctac catgcgttgc atgagaacag agccgaccat 3900
tccgcgccag ccgataaaac caacattttt catactcttc ctttttcaat attattgaag 3960
catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa 4020
Claims (10)
1. An expression plasmid for a bacterial asd gene balance lethal system, wherein the expression plasmid for the bacterial asd gene balance lethal system is a pCW1910 plasmid and comprises a Trc promoterA multiple cloning site, rrnB T1 terminator, rrnB T2 terminator, bom region, ori replication initiation site, β lactamase andasda gene; the nucleotide sequence of pCW1910 plasmid is
AATGAGCTGTTGACAATTAATCATCCGGCTCGTATAATGTGTGGGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGACCATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGAATTCGCAATTCCCGGGGATCCGTCGACCTGCAGCCAAGCTCCCAAGCTTTTGGCTGTTTTGGCGGATGAGAGAAGATTTTCAGCCTGATACAGATTAAATCAGAACGCAGAAGCGGTCTGATAAAACAGAATTTGCCTGGCGGCAGTAGCGCGGTGGTCCCACCTGACCCCATGCCGAACTCAGAAGTGAAACGCCGTAGCGCCGATGGTAGTGTGGGGTCTCCCCATGCGAGAGTAGGGAACTGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATCCGCCGGGAGCGGATTTGAACGTTGCGAAGCAACGGCCCGGAGGGTGGCGGGCAGGACGCCCGCCATAAACTGCCAGGCATCAAATTAAGCAGAAGGCCATCCTGACGGATGGCCTTTTTGCGTTTCTCACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCGCAGCCATGACCCAGTCACGTAGCGATAGCGGAGTGTATACTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGACTAGTTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTGCAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAACACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGACTAGTCTACGCCAACTGGCGCAGCATTCGACGCAGCGGCTCGGCGGCGCCCCATAACAACTGGTCGCCTACGGTAAACGCCGACAAGAACTCTGGCCCCATGTTCAGCTTACGCAGACGACCAACCGGCGTAGTCAACGTGCCGGTCACCGCCGCCGGGGTTAATTCGCGCATAGTGATATCACGATCGTTCGGCACCACTTTCGCCCACGGATTATGTGCCGCCAGCAGTTCTTCCACCGTCGGAATGGATACCTCTTTTTTCAGCTTGATGGTGAACGCCTGGCTGTGACAGCGCAGCGCGCCGACGCGCACGCACAAACCATCAACCGGAATCACAGAGGCAGTATTGAGAATCTTGTTGGTTTCCGCCTGGCCTTTCCACTCTTCGCGGCTCTGGCCGTTATCGAGCTGTTTGTCGATCCAGGGGATCAGGCTTCCCGCCAGCGGTACGCCAAAGTTATCAACCGGCAGCTCGCCGCTGCGGGTCAATGCCGTAACTTTGCGTTCAATATCAAGAATTGCGGAAGACGGCGTCGCCAGTTCATCGGCGACATGGCCATACAACTGACCCATCTGGGTTAACAGCTCGCGCATATGGCGCGCGCCGCCGCCGGAGGCGGCCTGATAGGTCGCGACGGATACCCAGTCAACGAGATTATGGGCAAAGAGACCGCCCAGCGACATCAACATCAGGCTAACGGTACAGTTACCGCCCACAAAGGTCTTCACGCCATTGTTCAGGCCGTCGGTAATCACGTCCTGGTTAACCGGGTCGAGAATAATAATGGCATCATCTTTCATGCGCAGCGTAGAAGCCGCGTCAATCCAGTAACCCTGCCATCCGCTTTCGCGCAGCTTTGGATAAATTTCGTTGGTATAATCGCCGCCCTGGCAGGTCACGATGATATCGAGCGCTTTTAGCGCATCCAGATCAAAAGCGTCCTGTAGCGTGCCGGTGGAGGTGTCGCCGAAGGTGGGCGCCGCCTGTCCAAACTGGGAGGTAGAAAAGAAAACAGGGCGAATAGCGTCGAAATCGCGCTCCTCTACCATGCGTTGCATGAGAACAGAGCCGACCATTCCGCGCCAGCCGATAAAACCAACATTTTTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCG。
2. The expression plasmid for a bacterial asd gene balanced lethal system according to claim 1, wherein the nucleotide sequence of the Trc promoter is the 10 th to 39 th positions of the nucleotide sequence of the pCW1910 plasmid.
3. The expression plasmid for balanced lethal system of bacterial asd gene as claimed in claim 1, wherein the nucleotide sequence of rrnB T1 terminator is 448-534 th site of pCW1910 plasmid nucleotide sequence;
the nucleotide sequence of the rrnB T2 terminator is 626-653 th of the nucleotide sequence of the pCW1910 plasmid.
4. The expression plasmid for bacterial asd gene balanced lethal system as claimed in claim 1, wherein the nucleotide sequence of the bom region is 764-904 of the nucleotide sequence of pCW1910 plasmid.
5. The expression plasmid for bacterial asd gene balanced lethal system according to claim 1, wherein the nucleotide sequence of ori replication initiation site is 1090-1678 th site of the nucleotide sequence of pCW1910 plasmid.
6. The expression plasmid according to claim 1, wherein the β -lactamase gene is present in both the head and tail endsSpeI restriction endonuclease site, the nucleotide sequence of which is 1849-2826 of the nucleotide sequence of pCW1910 plasmid.
7. The expression plasmid for balanced lethal system of bacterial asd genes as claimed in claim 6, wherein the nucleotide sequence of asd gene is 2827-3933 of the nucleotide sequence of pCW1910 plasmid.
8. Use of the expression plasmid for a bacterial asd gene balanced lethal system according to any one of claims 1 to 7, wherein said expression plasmid for a bacterial asd gene balanced lethal system is used for expressing green fluorescent protein in attenuated salmonella.
9. Use according to claim 8, characterized in that it is carried out in the following sequence of steps:
(I) gfp Gene amplification
Taking a plasmid containing a GFP gene as a template, taking GFP-F and GFP-R as primers, amplifying the GFP gene by adopting a PCR technology to obtain a PCR product, detecting the PCR product by using agarose gel electrophoresis, cutting a target strip, and purifying by using a gel recovery kit to obtain a GFP gene fragment;
GFP-F：5-AAGCTTATGGAGAGCGACGAGAGC-3
GFP-R：5-AAGCTTTCAGCGAGATCCGGTGGAGC-3
(II) pEASY-blunt-gfp construction
Connecting the amplified and purified gfp gene fragment to a pEASY-blunt cloning vector;
(III) construction of pCW1910-gfp
Using restriction endonuclease Hind III to cut pEASY-blunt-gfp and pCW1910 plasmid, recovering gfp gene fragment and pCW1910 plasmid, and connecting;
(IV) HX.DELTA.asd Strain construction
Preparing HX strain competent cells by using single HX strain colonies, adding pKD46 plasmid for transformation, screening HX/pKD46 positive colonies, performing electrotransfer by using pKD4 plasmid as a template and asd-F and asd-R as primers, performing PCR amplification on purified targeting gene fragments, eliminating pKD46 plasmid after screening positive colonies, performing electrotransformation by using pCP20 plasmid to eliminate pCP20 plasmid, and detecting to obtain HX delta asd strain;
asd-F：5’-AGGATACTGGCGCGCATACACAGCACATCTCTTTGCAGGAAAAAAACGCTGTGTAGGCTGGAGCTGCTTC-3’
asd-R：5’-TATCCGGCCTACAGAACCACACGCAGGCCCGATAAGCGCTGCAATAGCTATTAACGGCTGACATGGGAATTAG-3’
(V) pCW1910-gfp HX.DELTA.asd strain transformation
And transforming the pCW1910 plasmid connected with the gfp gene into an HX delta asd strain to obtain the HX delta asd/pCW1910-gfp strain.
10. The use according to claim 9, wherein the HX Δ asd/pCW1910-gfp strain has the accession number of CGMCC No.19579, is deposited in the china general microbiological culture collection center for a period of 2020, 04/15 days; the HX delta asd strain has the preservation number of CGMCC No.19578, is preserved in the China general microbiological culture Collection center of China Committee for culture Collection of microorganisms, and has the preservation time of 2020, 04, 15 days.
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